Mass spectrometers have become increasingly sophisticated, analytically powerful instruments offering well-established MS/MS methods for peptide sequencing and proteomics applications. However, despite the impressive array of high performance instruments, there remains a need for extending the flexibility of mass spectrometric strategies for targeted applications as well as enhancing both the selectivity and tunability. The objective of this proposal is the development of a new front-end method for manipulation of ions prior to introduction into the mass spectrometer, thus adding versatility to tandem mass spectrometry workflows. The strategy is based on a dual spray reactor that allows intersection of analyte ions with a second population of reagent ions or molecules, promoting gaseous ion-molecule and ion-ion reactions at atmospheric pressure. The spray reactor will facilitate on-line derivatization of analyte ions, manipulation of charge states based on acid-base chemistry, and addition of internal standards for quantitative and calibration purposes. For bottom-up proteomics applications, the dual spray reactor will expand the flexibility of activation methods by allowing a convenient means for manipulation of charge states and chemical derivatization. Morever, the capabilities of promoting selective reactions to add cleavable reporter tags, to attach chromophores, or to conveniently add internal standards to improve precision, comprise some of the ways that front-end control over ion chemistry offers an added dimension of experimental flexibility. An array of ion-ion and ion-molecule reactions will be implemented via the dual spray reactor including: (i) manipulation of charge states via acid/base chemistry and addition of ionizable groups, (ii) selective reactivity based on functional group, (iii) addition of chromophores to enhance photodissociation, (iv) attachment of reporter tags to facilitate tracking ions via SRM scans (data-dependent mode), and (v) infusion of reference standard to improve run-to-run precision. Adaptation for targeted bottom-up proteomics applications offers a new paradigm for extending the capabilities of existing high performance mass spectrometers and affords significant potential for extending the tunability, flexibility, and versatility of the standard ESI-MS platform for proteomics applications. 1